Project Description: Thyroid cancer incidence is rapidly increasing in the United States. Veterans are at even higher risk of developing thyroid cancer in their lifetime due to the increased potential of radiation exposure in the armed services. Furthermore, veterans who suffered from thyroid cancer self-reported a higher prevalence of Agent Orange exposure, increasing the relevance of this malignancy to the VA population. Despite the use of a range of combinations of treatments, ATC exhibits a dismal prognosis with a median survival of < six months. ATC tumors possess a greater mutation burden than all other forms of thyroid cancer which impart significant growth benefit and high metastatic potential Therefore, it is important to identify proteins that function at ?nodal points? of different signaling pathways implicated in ATC, and thus could represent ATC ?Achilles Heel.? The CTNNB1 (?-catenin) is an effector molecule of Wnt signaling which is critical for epithelial-mesenchymal transition (EMT) required for metastasis. PI3K/Akt/GSK3? signaling is hyperactive in ATC due to mutations. This can enhance ?-catenin activity and also phosphorylate Map kinase Activating Death Domain-containing protein (MADD) and contribute to its pro-survival function. pMADD renders ATC cells resistant to apoptosis. Thus in ATC, pMADD and ?-catenin can serve as two potential ?nodal points.? Most importantly, our recent novel findings have shown that MADD knockdown can significantly inhibit TNF? mediated activation of ?-catenin signaling by preventing pERK activation and consequent pGSK3? activation. Lack of GSK3? phosphorylation by ERK, facilitated ubiquitination of ?-catenin leading to its degradation and resultant blockage of EMT activation. Furthermore, intra-tumoral administration of MADD siRNA significantly reduced orthotpic ATC tumor growth and lung metastasis in treated mice. Therefore, MADD is a potential therapeutic target in ATC either alone or in combination with Wnt/?-catenin inhibitors. Based on a very strong scientific premise, we hypothesize that MADD down modulation can be effective in inhibiting growth and overcoming resistance to drugs targeting hyperactive MAPK, PI3K/Akt and Wnt/?-catenin signaling, which are hallmarks of ATC. To address this, in aim-1, we will functionally characterize the impact of CRISPR/CAS9 mediated MADD gene knock-out in ATC cells on Wnt signaling in vitro and ex vivo; in aim-2, we will determine the impact of down-modulating MADD expression, and MAPK and PI3K/Akt signaling pathways, on Wnt signaling; and in aim-3, we will test the in vivo therapeutic efficacy of combination treatment with MADD knockdown in orthotopic and spontaneous mouse models of ATC. ATC disproportionately accounts for the majority of thyroid cancer-related deaths. Cancer cell-specific expression of MADD, its role in activating several key signaling pathways, and its ability to act as a pro-survival factor and promote metastasis in ATC makes it an ideal target for therapeutic development. Importantly, MADD deletion had no apparent effect on normal cells. Thus the proposed novel studies are highly relevant to veterans.